Pleomorphic adenoma gene1 in reproduction and implication for embryonic survival in cattle: a review

Abstract The pleomorphic adenoma gene1 (PLAG1) encodes a DNA-binding, C2H2 zinc-finger protein which acts as a transcription factor that regulates the expression of diverse genes across different organs and tissues; hence, the name pleomorphic. Rearrangements of the PLAG1 gene, and/or overexpression, are associated with benign tumors and cancers in a variety of tissues. This is best described for pleomorphic adenoma of the salivary glands in humans. The most notable expression of PLAG1 occurs during embryonic and fetal development, with lesser expression after birth. Evidence has accumulated of a role for PLAG1 protein in normal early embryonic development and placentation in mammals. PLAG1 protein influences the expression of the ike growth factor 2 (IGF2) gene and production of IGF2 protein. IGF2 is an important mitogen in ovarian follicles/oocytes, embryos, and fetuses. The PLAG1-IGF2 axis, therefore, provides one pathway whereby PLAG1 protein can influence embryonic survival and pregnancy. PLAG1 also influences over 1,000 other genes in embryos including those associated with ribosomal assembly and proteins. Brahman (Bos indicus) heifers homozygous for the PLAG1 variant, rs109815800 (G > T), show greater fertility than contemporary heifers with either one, or no copy, of the variant. Greater fertility in heifers homozygous for rs109815800 could be the result of early puberty and/or greater embryonic survival. The present review first looks at the broader roles of the PLAG1 gene and PLAG1 protein and then focuses on the emerging role of PLAG1/PLAG1 in embryonic development and pregnancy. A deeper understanding of factors which influence embryonic development is required for the next transformational increase in embryonic survival and successful pregnancy for both in vivo and in vitro derived embryos in cattle.


Introduction
The major cause of reproductive loss in cattle is the failure of embryos to progress to implantation and pregnancy.Fertilization rates in both beef and dairy cattle are in the order of 85% to 100%; however, only 40% to 60% of embryos establish a pregnancy (Diskin et al., 2016;Lockhart et al., 2023).In recent reviews, we have argued that the next transformational change in reproductive efficiency will require a deeper understanding of the biology of early embryo development in cattle (D'Occhio et al., 2019b(D'Occhio et al., , 2020a, b;, b;Campanile et al., 2021).This applies to both natural mating and assisted reproduction.A critically important feature of early embryo development is the dialogue between embryo and uterus in the period before embryo attachment and during implantation (Hantak et al., 2014;Rizos et al., 2017;Sponchiado et al., 2017Sponchiado et al., , 2019Sponchiado et al., , 2020;;Aguilera et al., 2022;Binelli et al., 2022;Cajas et al., 2022;Tesfaye et al., 2022).Factors involved in embryo-uterine communication include the transforming ß superfamily (D'Occhio et al., 2020a), cell-cell adhesion molecules (D'Occhio et al., 2019b), kisspeptin (D'Occhio et al., 2020b) and immune factors (Campanile et al., 2021), among others.Our reviews, and those of others, have noted the complexity of events associated with early embryo development, attachment of the conceptus to the uterine epithelium, and implantation.The reviews have identified major gaps in our understanding of early embryo development in cattle.The gaps in knowledge largely explain the relatively modest progress over the past 40 yr in reducing high embryo loss in cattle.High embryo loss applies to both in vivo and in vitro derived embryos.Embryo loss is comparable after natural mating, artificial insemination, or embryo transfer (Hansen, 2020).The transfer of a bovine embryo to a recipient at day 7 of development avoids the relatively large loss of embryos that occurs in the first 7 d after fertilization.However, there is still considerable loss between the transfer on day 7, and day 21, when embryo attachment has commenced (Hansen, 2020).Therefore, the transfer of a bovine embryo on day 7 of early development does not overcome all the embryo losses in cattle that occur before implantation.
The approach adopted in the present review is to first provide a general background on the PLAG1 gene and PLAG1 protein.We then consider relationships between PLAG1 polymorphisms and phenotypes in cattle.This is followed by a focus on the role of PLAG1/PLAG1 in early embryonic development.In keeping with our earlier reviews, this review seeks to build awareness of the complex biology of embryonic development.Our consistent argument has been that a deeper understanding is needed of the factors that impact early embryo development before a meaningful transformational change can be made in the efficiency of both natural mating and assisted reproduction in cattle.

Discovery of PLAG1 Gene and PLAG1 Protein
The PLAG1 gene and PLAG1 protein were described from 1997 to 1998 (Table 1).The seminal report showed the PLAG1 gene to be associated with a chromosome translocation at 8q12 that was linked with pleomorphic adenomas of the salivary glands in humans (Kas et al. 1997a, b).The same laboratory described two related human proteins, PLAGL1 and PLAGL2.The protein PLAGL2 also binds to DNA and has similar properties as PLAG1 protein (Kas et al. 1998).The PLAG1/PLAG1 family members were subsequently assigned various names based on the association of PLAG1 mutations with different phenotypes in different species (Table 1).In the absence of PLAG1 gene rearrangement, and/or overexpression of PLAG1, PLAG1 protein can have antiproliferative activity and tumor suppression.Hence, the regulated expression of PLAG1 is associated with normal cellular function in different tissues, while overexpression is linked with benign tumors and malignancies (Zatkova et al., 2004).Overexpression of PLAG1 leads to overproduction of PLAG1, rather than changes in the structure of PLAG1 protein.PLAG1 stimulates the IGF2 gene and excess production of IGF2 is considered one mechanism linked to tumors and cancers (Voz et al., 2000(Voz et al., , 2004;;Zatkova et al., 2004;Akhtar et al., 2012).
In humans, the PLAG1 gene comprises 6 exons and 5 introns.PLAG1 has yet to be fully described in cattle and is presently thought to comprise 3 introns and 4 exons (Van Dyck et al., 2007; Figure 2).In cattle, a 19-base pair inser-tion/deletion (19-bp indel) at Exon 1, and single-nucleotide polymorphisms at Exons 3 and 4, are associated with growth, stature, and carcass traits (Karim et al. 2011;Littlejohn et al. 2011;Zhong et al., 2019; Figure 2).PLAG1 mutations were also associated with age at puberty and circulating levels of IGF1 in heifers (Fortes et al., 2013).PLAG1 is located within the same quantitative trait loci as the coiled-coil-helix-coiledcoil-helix domain containing 7 (CHCHD7) gene, which is also associated with growth and stature in several species including cattle (Li et al., 2020a;Xu et al., 2020).Both genes share the same bi-directional promoter and SNPs known to influence the transcriptional activity of the promoter impact the expression of PLAG1 and CHCHD7 (Karim et al., 2011;Fink et al., 2017; Figure 2).PLAG1 protein is comprised of three regions with distinct functions: a region with nuclear translocation signals for the transfer of PLAG1 to the nucleus; C 2 H 2 -like zinc-finger domains that interact with DNA to influence transcription; a serine-rich region that has transcriptional activation activity (Braem et al., 2002;Hensen et al., 2002; Figure 2).

Puberty
Age at puberty is a highly important trait which is linked to lifetime fertility in female cattle (Hawken et al., 2012;Wathes et al., 2014;D'Occhio et al., 2019a).Mutations on chromosome 14 (BTA14), in proximity to PLAG1, were reported to be associated with puberty in Zebu (Bos indicus) heifers including Brahman (Hawken et al., 2012;Fortes et al., 2013) and Nellore (Mota et al., 2020).Heifers with delayed puberty linked to various PLAG1 mutations are heavier at puberty.Over 36 yr, we have subjected a herd of Brahman (Bos indicus) females to uncompromising selection for fertility (Collins Belah Valley [CBV] Brahman, Belah Valley Cattle Station, Marlborough, Central Queensland, Australia).Females remain in this herd only if they conceive, wean a calf, and reconceive in successive years starting with their first mating (Collins A. Snr., J. E. Kinder, and M. J. D'Occhio, unpublished).Days-to-calving (DTC), defined as the number of days from the start of mating to subsequent calving, is the most important measure of fertility in Brahman and the key driver of profit in beef production.Herd records are used to calculate estimates of genetic differences between animals for DTC and these are expressed as estimated breeding value (EBV) or estimated progeny difference.Female cattle with a low DTC EBV show early puberty as heifers and resume cyclic ovarian function sooner after calving.The DTC EBV for the CBV Brahman herd is −16.8 d compared with the Australian Brahman breed average DTC EBV of −3.2 d.The latter demonstrates a strong genetic component for high fertility of the CBV Brahman herd.It was recently shown that maiden heifers in the CBV Brahman herd that were homozygous for the PLAG1 variant rs109815800 (G > T) conceived earlier and had greater fertility than contemporary heifers with either one or no copies of the variant (Engle & Hayes, 2022).Heifers with two copies of the variant had a smaller stature than heifers with one or no copies (Engle & Hayes, 2022).
Mouse embryos lacking maternal PLAG1 transitioned slowly from the 2-to 4-cell stage of development (Madissoon et al., 2019).Embryos that transition through early cell divisions in a timely manner have a greater likelihood of surviving and establishing a pregnancy.In mice that lacked maternal PLAG1 the gene was expressed ectopically from the paternal allele earlier than would otherwise occur (Madissoon et al., 2019).MicroRNAs (miRNAs) have been implicated in the function of PLAG1/PLAG1 in early development (Maccani & Marsit, 2011;Kochhar et al., 2021).For example, miRNA-141 downregulates PLAG1 translation which is associated with fetal growth retardation (Tang et al., 2013).Based on the relationship between PLAG1 and expression of the IGF2 gene discussed above, it was concluded that miRNA-141 downregulation of PLAG1 results in reduced IGF2, and suppressed fetal growth (Varrault et al., 2006;Tang et al., 2013;Saha et al., 2015).There is a lack of information on the specific localization of PLAG1 expression in the embryos and uterus and this is an area that warrants investigation.

PLAG1/PLAG1 and Embryos Survival in Cattle
As noted above, the failure of embryos to progress to implantation and pregnancy is the major cause of reproductive loss in cattle.A deeper understanding of the factors which support embryonic development, attachment, and implantation, is key to improving embryo survival and achieving a transformational increase in reproductive success in female cattle.The factors are both genetic and non-genetic, although these are clearly interrelated.As noted above, Brahman (Bos indicus) heifers homozygous for the PLAG1 variant, rs109815800 (G > T), show greater fertility than contemporary heifers with either one or no copy of the variant.Greater fertility in heifers homozygous for rs109815800 could be due to an earlier age at puberty and/or an increased propensity for embryo survival.The latter would mean that homozygous heifers require fewer matings to achieve pregnancy; typical embryo loss in cattle is in the order of 40% to 60%.Another PLAG1 variant, rs109231213, appears to be associated with central mechanisms of puberty in heifers (Fortes et al., 2013(Fortes et al., , 2016;;DeAtley et al., 2018).Based on the information provided in this review, it is plausible that PLAG1/PLAG1 have a role in embryonic development and survival in cattle.This is supported by the important roles of IGF2 in follicles/oocytes, embryos, and fetuses, and the regulation of IGF2/IGF2 by PLAG1.A role in central mechanisms associated with puberty in cattle is also plausible.
Notwithstanding the body of evidence that links PLAG1/ PLAG1 with IGF2 and embryonic development and reproduction generally, it is noted that some of the relationships in this review could be considered associations and further research is needed to demonstrate additional cause-and-effect relationships.

Summary
The present review has looked at the emerging roles of PLAG1/ PLAG1 in embryonic development, placentation, and fetal growth.The most notable expression of PLAG1 occurs during embryonic and fetal development, with lesser expression after birth.Overexpression of PLAG1 is associated with the large calf syndrome in cattle and under-expression is linked to fetal growth restriction in cattle and humans.The overexpression of PLAG1 later in life is typically associated with the formation of solid tumors and cancers.Hence, the expression of PLAG1 is finely balanced, and disruption in expression at different stages in life shifts PLAG1 from having beneficial effects to adverse outcomes.PLAG1/PLAG1 influence the expression of the IGF2 gene, and the IGF2 protein is an important mitogen in reproduction.The PLAG1-IGF2 axis, therefore, provides a mechanistic basis for an effect of PLAG1 on ovarian follicles/ oocytes, embryos, and fetuses.Our own work involving the selection of Brahman (Bos indicus) female cattle for fertility over a period of 35 yr has led to a herd in which heifers homozygous for the PLAG1 variant, rs109815800, have greater fertility than contemporary heifers with either one or no copy of the variant (Collins A. Snr, J. E. Kinder, B. J. Hayes, and M. J. D'Occhio, unpublished).PLAG1/PLAG1 would therefore appear to have important roles in embryonic development and pregnancy in cattle similar to other mammals.

Figure 1 .
Figure 1.Zygotic genome activation in cattle.PLAG1 is maternally imprinted and PLAG1 protein derived from paternally expressed PLAG1 could potentially be present in embryos from the 2 to 4 cell stage.

Figure 2 .
Figure 2. The putative structure of the PLAG1 gene in cattle and variants of PLAG1 associated with different phenotypes.Indel, insertion/deletion; SNPs, single-nucleotide polymorphisms (top); the common bi-directional promoter of the PLAG1 and CHCHD7 genes.QTLs/SNPs in the promoter influence the transcriptional activity of PLAG1 and CHCHD7 and phenotypes in cattle including growth and stature.QTLs, quantitative trait loci; SNPs, single-nucleotide polymorphisms (middle); and the structure of PLAG1 protein and domains associated with translocation to the nucleus and binding to DNA.PLAG1 typically binds to the promoter of target genes to influence transcription (bottom).

Figure 3 .
Figure 3. Insulin-like growth factor 2 (IGF2) is produced by the fetus and placenta and has both local and reciprocal action between the fetus and placenta.IGF2 can bind to both IGF1 and IGF2 receptors on target cells.

Figure 4 .
Figure 4. Insulin-like growth factor 2 (IGF2) is produced by oocytes and granulosa cells of follicles and has a local and reciprocal action in oocytes and follicles.IGF2 is an important mitogen and can bind to both IGF1 and IGF2 receptors at target cells.The IGF2 gene is influenced by PLAG1 protein which provides a mechanism for PLAG1 to be associated with oocyte and follicular function.

Table 1 .
Discovery of the pleomorphic adenoma gene (PLAG1) family members